Department of Genetics and Biotechnology, St. Petersburg State University, St. Petersburg, Russia.
Institute of Biology, Irkutsk State University, Irkutsk, Russia.
Prion. 2020 Dec;14(1):118-128. doi: 10.1080/19336896.2020.1751574.
Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was proposed by Vitaly V. Kushnirov in the Michael D. Ter-Avanesyan's laboratory as a method to compare sizes of amyloid aggregates. Currently, this method is widely used for amyloid investigation, but mostly as a qualitative approach. In this work, we assessed the possibilities and limitations of the quantitative analysis of amyloid aggregate size distribution using SDD-AGE results. For this purpose, we used aggregates of two well-characterized yeast amyloid-forming proteins, Sup35 and Rnq1, and developed a protocol to standardize image analysis and process the result. A detailed investigation of factors that may affect the results of SDD-AGE revealed that both the cell lysis method and electrophoresis conditions can substantially affect the estimation of aggregate size. Despite this, quantitative analysis of SDD-AGE results is possible when one needs to estimate and compare the size of aggregates on the same gel, or even in different experiments, if the experimental conditions are tightly controlled and additional standards are used.
半变性去污剂琼脂糖凝胶电泳（SDD-AGE）由 Vitaly V. Kushnirov 在 Michael D. Ter-Avanesyan 的实验室提出，作为比较淀粉样物聚集物大小的方法。目前，该方法广泛用于淀粉样物的研究，但主要是作为一种定性方法。在这项工作中，我们评估了使用 SDD-AGE 结果进行淀粉样物聚集物大小分布定量分析的可能性和局限性。为此，我们使用了两种经过充分表征的酵母淀粉样形成蛋白 Sup35 和 Rnq1 的聚集物，并开发了一种标准化图像分析和处理结果的方案。对可能影响 SDD-AGE 结果的因素进行了详细研究，结果表明，细胞裂解方法和电泳条件都可能会极大地影响聚集物大小的估计。尽管如此，如果需要在同一张凝胶上估计和比较聚集物的大小，或者在实验条件得到严格控制且使用了额外的标准时，在不同的实验中进行 SDD-AGE 结果的定量分析仍然是可行的。
